Guanylate cyclase has been purified 2000- to 5000-fold from rat liver supernatant. The highest specific activity was 120 nmol of cGMP/min/mg protein. Analytical gel electrophoresis revealed one major protein band with which the activity associated and one or two minor band(s). The pure enzyme exhibited classical Michaelis-Menten kinetics in the presence of activator fraction which was not retained by DEAE-Biogel during the purification of guanylate cyclase. In its absence, the enzyme activity was not proportional to protein concentration and showed anomalous kinetics with GTP. The enzyme is free of GTPase adenylate cyclase, and phosphodiesterase. Crude guinea pig liver guanylate cyclase is activated by Co 2 ion but not by other metallic ions or cobaltic compounds. After partial purification of the enzyme, Co+2 ion no longer activates but addition of a heat-stable low molecular weight fraction from guinea pig liver restores sensitivity to Co+2 ion. The rat liver enzyme is not activated by Co 2 ion, even when this fraction is added. Identification of the factor(s) responsible for Co+2 ion activation is in progress.